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Originally published In Press as doi:10.1074/jbc.M411042200 on November 4, 2004

J. Biol. Chem., Vol. 280, Issue 8, 7206-7217, February 25, 2005
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Regional Specificity of Human ether-a'-go-go-related Gene Channel Activation and Inactivation Gating*{boxs}

David R. Piper{ddagger}§, William A. Hinz{ddagger}§, Chandra K. Tallurri{ddagger}§, Michael C. Sanguinetti{ddagger}§, and Martin Tristani-Firouzi§||

From the {ddagger}Department of Physiology, §Nora Eccles Harrison Cardiovascular Research and Training Institute and the ||Department of Pediatrics, University of Utah, Salt Lake City, Utah 84112

Received for publication, September 27, 2004 , and in revised form, October 27, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Slow activation and rapid C-type inactivation produce inward rectification of the current-voltage relationship for human ether-a'-go-go-related gene (hERG) channels. To characterize the voltage sensor movement associated with hERG activation and inactivation, we performed an Ala scan of the 32 amino acids (Gly514-Tyr545) that comprise the S4 domain and the flanking S3–S4 and S4–S5 linkers. Gating and ionic currents of wild-type and mutant channels were measured using cut-open oocyte Vaseline gap and two microelectrode voltage clamp techniques to determine the voltage dependence of charge movement, activation, and inactivation. Mapping the position of the charge-perturbing mutations (defined as |{Delta}{Delta}G| > 1.0 kcal/mol) on a three-dimensional S4 homology model revealed a spiral pattern. As expected, mutation of these residues also altered activation. However, mutation of residues in the S3–S4 and S4–S5 linkers and the C-terminal end of S4 perturbed activation (|{Delta}{Delta}G| > 1.0 kcal/mol) without altering charge movement, suggesting that the native residues in these regions couple S4 movement to the opening of the activation gate or stabilize the open or closed state of the channel. Finally, mutation of a distinct set of residues impacted inactivation and mapped to a single face of the S4 helix that was devoid of activation-perturbing residues. These results define regions on the S4 voltage sensor that contribute differentially to hERG activation and inactivation gating.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The human ether-a'-go-go-related gene (hERG)1 channels are primarily expressed in the brain and the heart (1) but are also up-regulated in tumors from a variety of tissues (2). In the heart, hERG channels conduct the rapidly activating, delayed rectifier potassium current, IKr (3, 4). Unlike most voltage-gated K+ (Kv) channels, the fully activated current-voltage relationship of hERG channels exhibits inward rectification, a property that limits efflux of K+ during the plateau phase of the cardiac action potential.

Inward rectification of hERG channels results from a rapid and voltage-dependent C-type inactivation that proceeds at a rate much faster than activation. At membrane potentials between -20 and +20 mV, channels activate over hundreds of milliseconds but inactivate in milliseconds (5–7). The voltage dependence of hERG inactivation is shifted nearly -65 mV relative to channel activation (V0.5INACT = -85 mV, V0.5ACT = -20 mV). A single mutation (S631A) in the P-loop of the outer pore causes a +100 mV shift in the voltage dependence of inactivation, but no shift in activation compared with wild-type (WT) channels (6, 8). Furthermore, external cations have differential effects on the voltage dependence of activation and inactivation (9–11). Together, these findings led to the suggestion that distinct voltage-sensing mechanisms underlie activation and inactivation gating in hERG channels.

The highly charged S4 transmembrane helix functions as the primary voltage sensor in voltage-gated channels. Neutralization of basic S4 residues shifts the voltage dependence of Shaker channel activation (12) and reduces gating current (13–15). Moreover, hydrophobic residues surrounding the charged S4 residues are also important in voltage sensing (16–19). Although controversy exists regarding the specific details of how the S4 helix responds to changes in membrane potential, the bulk of evidence indicates that its physical movement generates gating currents that precede channel opening or closing (20–23).

Voltage sensor operation in hERG channels has been assessed by voltage clamp fluorometry (24) and by measurement of gating currents (25). Both studies revealed fast and slow components that differed nearly 100-fold in their kinetics. The majority of the gating charge displacement was slow and associated with channel activation. The fast gating component, although kinetically similar to inactivation, had a midpoint nearly 100 mV more positive than the midpoint of hERG inactivation (24, 25) and likely represents early independent transitions in the activation pathway.

What determines the voltage sensitivity of fast inactivation? To address this question, we measured gating currents from the inactivation-deficient S631A hERG channel. Despite dramatic differences in inactivation, we did not detect any difference in gating currents between WT and S631A hERG channels (25). Moreover, no differences in fluorescence were detected between WT hERG and a channel with two mutations (G628C/S631C) in the P-loop that completely removed inactivation (24). These studies suggest that mutations in the outer pore uncouple voltage sensor movement from the final rearrangements in the pore domain that close the inactivation gate.

To further characterize the voltage sensor movement associated with hERG activation and inactivation, we performed an Ala scan of the S4 domain and the flanking S3–S4 and S4–S5 linkers to test the hypothesis that individual substitutions might differentially alter the equilibrium between the resting and activated states of the voltage sensor, the closed and open activation gate, or the open and inactivated pore. Here we report that Ala substitutions that perturbed charge movement localized to a spiral thread along the S4 helix. Mutations that perturbed activation were scattered throughout the scanned region, with the exception of one helical face of S4. Mutations that perturbed inactivation localized to the helical face of S4 that was devoid of activation-perturbing residues. Taken together, these results suggest that the hERG S4 acts as a voltage sensor for activation and inactivation but that distinct regions of S4 contribute differentially to each process, likely through specific and state-dependent inter-domain contacts.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Molecular Biology and Oocyte Injection—Site-directed mutagenesis (26) of hERG, in vitro synthesis of cRNA, isolation and injection of Xenopus laevis oocytes were performed as previously described (27, 28). Oocytes were injected with 2 ng of cRNA to record ionic current or 15 ng cRNA to record gating currents. Injected oocytes were incubated at 17–19 °C for 2–4 days before use in voltage clamp experiments.

Electrophysiology—To estimate activation parameters, ionic currents were measured with a TEV-200 amplifier (Dagan Corp.) using the two-microelectrode voltage clamp technique (28). Currents were filtered at 1 kHz with an eight-pole Bessel filter and then digitized at 2 kHz. To estimate charge movement and inactivation parameters, gating and ionic currents were measured with a CA-1B amplifier (Dagan Corp.) using the cut-open Vaseline gap (COVG) voltage clamp technique (29). Currents were filtered at 5 kHz with an eight-pole Bessel filter, and then digitized at 20 kHz. Voltage commands were generated with a personal computer using pCLAMP8 software and a DigiData 1322 analog to digital interface (Axon Instruments).

Microelectrodes were pulled from borosilicate glass capillary tubes (WPI) to obtain resistances of 0.7–2.0 M{Omega} for use in two-microelectrode voltage clamp and 0.1–0.5 M{Omega} for use in COVG when filled with 3 M KCl. All recordings were performed at 22–24 °C. For two-microelectrode voltage clamp recordings, the extracellular solution contained (in mM): 96 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, pH 7.6. The recording chamber was perfused at a rate of ~1 ml/min. The same extracellular solution was used for the top and guard chambers in COVG recordings. The intracellular solution in the bottom COVG chamber contained (in mM): 120 K-glutamate, 10 HEPES, pH 7.0. For gating current recordings, the extracellular solution consisted of (in mM): 120 tetraethylammonium hydroxide, 120 MES, 2 Ca-MES, 10 HEPES, pH 7.4. The intracellular solution contained (in mM) 120 tetraethylammonium hydroxide, 120 MES, 10 HEPES, pH 7.4. All chemicals were purchased from Sigma except MK-499, which was a gift from Merck & Co.

For COVG recordings, the oocyte was permeabilized by adding 0.1–0.3% saponin in 120 potassium glutamate, 10 HEPES (pH 7.0) to the lower chamber for 2 min. For gating current measurements, intracellular K+ was reduced by clamping the membrane to -10 mV for 20–30 min. To eliminate hERG ionic currents, the specific blocker MK-499 (50 µM) (30) was added to the external and internal solutions after depletion of intracellular K+. MK-499 did not appreciably alter the properties of hERG gating current (25), but was required to effectively eliminate residual ionic current during prolonged step depolarizations. Linear leak and capacitance currents were compensated by analog circuitry and subtracted on-line using a p/-8 protocol (31) from a holding potential (HP) of -110 mV. Nonlinear transient currents were not detected in uninjected oocytes under these conditions, indicating that gating currents detected in hERG cRNA-injected oocytes were not contaminated by constitutively expressed channels.

Data Analysis and Calculation of Free Energy Parameters—The impact of individual residues on charge movement, activation, and inactivation was assessed in relative isolation by performing a systematic Ala scan of the S4 helix and the S3–S4 and S4–S5 linkers (Gly516-Tyr545). To quantify the energetic perturbation produced by the individual mutations, we calculated the free energy difference ({Delta}G) of three gating transitions in WT channels and compared these values to the energy difference induced by Ala substitution. The difference in {Delta}G between WT and mutant channels ({Delta}{Delta}G) reflects the perturbation in a gating transition produced by the Ala substitution. Inherent in this analysis is the assumption that gating transitions can be simplified as two state transitions (e.g. closed to open). We examined the effect of Ala substitutions on three simplified equilibrium distributions reflecting the resting and activated voltage sensor, the closed and open activation gate, and the closed and open inactivation gate.

To determine the equilibrium distribution associated with the resting and activated voltage sensor, intramembrane charge (Q) displacement was calculated by integrating the off-gating current (IgOFF) at HP = -110 mV elicited after 300-ms depolarizing step changes in membrane voltage (-100 to +40 mV, 10-mV increments). The more negative Q-V relationship for D540A hERG channels necessitated a HP of -140 mV. Normalized values of Q (Qrel) were plotted versus test potential and the relationship fitted to a Boltzmann function, Equation 1,

(Eq. 1)
where Qmax is the maximum charge moved, Vt is the test potential, V0.5 is the voltage required to elicit 0.5*Qmax, z is the effective valence, F is Faraday's constant, R is the ideal gas constant, and T is temperature in degrees Kelvin.

To determine the closed-open channel equilibrium, the relative conductance-voltage (grel-V) relationship was determined by an analysis of deactivating ionic currents at -70 mV. Peak values were estimated by fitting deactivating currents to a bi-exponential function and extrapolating back to the moment of repolarization. The fraction of maximal current activated by the depolarizing step (I/Imax) was determined and used to construct the grel-V relationships.

The open-inactivated equilibrium (Iinact-V) was determined using a three-pulse protocol (5). First, hERG channels were activated and inactivated by a 300-ms pulse (Vpre) to +40 mV, then channels were recovered from inactivation by a 10-ms hyperpolarizing interpulse (Vinter) that was varied from -120 to +40 mV. Finally, the magnitude of the recovered currents was determined by a third test pulse (Vtest = +40 mV). For R531A mutant channels, Vpre and Vtest was +100 mV, because the V0.5 for activation was shifted to depolarized potentials. Iinact-V and grel-V curves were fitted with Boltzmann functions to estimate V0.5 and z.

V0.5 and z values were used to estimate the {Delta}G between the resting and activated voltage sensor, the closed and open activation gate, and the closed and open inactivation gate for WT and each mutation by Equations 2 and 3 as follows.

(Eq. 2)

(Eq. 3)
Perturbations in the {Delta}G ({Delta}{Delta}G) were estimated for each mutation by Equations 4 and 5.

(Eq. 4)

(Eq. 5)
Data are expressed as mean ± S.E. (n = number of oocytes). Data plots, Boltzmann fits, {Delta}{Delta}G calculations, and histogram analyses were performed with Origin 7.0 (OriginLabs Corp.) and Excel (Microsoft). The absolute {Delta}{Delta}G values were clustered using average linkage of Euclidean distances with Cluster (www.lohninger.com).

Linear color scales were constructed from the estimated perturbations (|{Delta}{Delta}G|) for charge movement, activation, and inactivation. For a given equilibrium distribution, its representative color was set to maximal and the remaining colors were reduced linearly with respect to |{Delta}{Delta}G|. For example, for the {Delta}{Delta}G for activation, green was set to 239 while red and blue were reduced according to the magnitude of perturbation using Equations 6 and 7,

(Eq. 6)

(Eq. 7)
where ScaleFactor = 2 (charge movement), 2 (activation), and 1 (inactivation).

An arbitrary scale factor was used to provide the most dynamic range of color possible for a given distribution, and a helical surface representation of S4 was colored according to the [R, G, B] values for each residue.

Alignments and Structural Homology Models—An alignment of the Shaker, EAG (ether-a'-go-go), KvAP, and hERG channels was constructed using ClustalW (32) with minor manual adjustments. The S4 homology model was based on the KvAP S1–S4 crystal structure (33). A homology model of a single hERG subunit was based on a model of the Shaker channel (34). Models were built with ModelerV6.2 (35), and images were constructed with DeepView (36) and the Persistence of Vision Raytracer (www.povray.org).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Ala Scanning—The putative secondary structural elements and the scanned region of a hERG channel subunit are depicted in Fig. 1. Also shown is an alignment with the homologous regions of EAG, Shaker, Kv2.1, and KvAP channels. Thirty-two residues (Gly514-Tyr545), including the S3–S4 linker through the S4–S5 linker, were mutated to Ala. Two mutant channels (K525A and L529A) did not express sufficiently to allow measurement of ionic or gating currents and were excluded from further study. Four other mutant channels did not express at levels high enough to permit gating current recordings (G514A, G516A, A527V, and R528A), but none of these perturbed the grel-V relationship (Tables I and II).



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  		FIG. 1.
Topology of hERG channels and alignment with related Kv channels. Top: putative secondary structure of one hERG {alpha}-subunit diagrams the six transmembrane {alpha}-helices (S1–S6) and the re-entrant pore helix (P). The region chosen for Ala-scanning is boxed and includes the S3–S4 linker, S4, and the S4–S5 linker. Bottom: the scanned region of hERG is aligned with the related Drosophila EAG, Shaker, Kv2.1, and KvAP channel sequences. The scanned region is boxed and connected to illustration by dotted lines. The black box represents the approximate limits of the S4 helix. Conserved residues are shaded gray, and identical residues are shaded black.

 


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  		TABLE I
Charge movement parameters for hERG channels derived from Qrel-V relationships fitted with Boltzmann functions

 


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  		TABLE II
Activation parameters for hERG channels derived from grel-V relationships fitted with Boltzmann functions

 
Specific Substitutions in S4 Perturbed Charge Movement— On-gating current (IgON) of WT channels is composed of a fast component (fast IgON) followed by a slow, smaller amplitude component (slow IgON) that represents ~90% of the total gating charge (25). These distinct components of IgON are clearly distinguishable during a pulse to +20 mV for WT channels (top panel of Fig. 2A). Although the mutations had a variable effect on the kinetics of gating current, the IgON of all channels could also be readily separated into fast and slow components. R531A, V535A, and D540A mutations had the most dramatic effects on the kinetics and voltage dependence of gating currents. R531A gating current was only detected at potentials ≥ -20 mV, and no slow component was evident at +20 mV (Fig. 2B), indicating a large positive shift in the voltage dependence of charge movement. V535A (Fig. 2C) and especially D540A (Fig. 2D) accelerated the rate of the slow IgON and IgOFF.



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  		FIG. 2.
Gating currents of WT and S4 Ala-substituted mutant channels. A–D, representative gating currents elicited from WT hERG (A), R531A (B), V535A (C), and D540A (D) mutant channels. The membrane potential was stepped from a HP of -110 mV to a range of test potentials for 300 ms and then returned to HP. Traces recorded from steps to +20, -20, and -60 mV are shown in each panel. Current calibration bar is 50 nA for panels A–C and 61 nA for panel D.

 
The V0.5 and z for the Qrel-V relationship of WT channels was -22 ± 1.9 mV and 2.2 ± 0.1 e0 (n = 8). Qrel-V relationships determined for mutant channels that caused the greatest shifts in V0.5 are plotted in Fig. 3A. V535A and A536V shifted the relationship to the left and L524A and R531A shifted it to the right. The Qrel-V relationship for D540A channels was unusual and best described as the sum of two Boltzmann functions. The more positive component overlapped with WT, but the more negative component was shifted by 62 mV in the hyperpolarized direction.



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  		FIG. 3.
Effects of Ala substitutions on the voltage dependence of gating charge movement. A, Qrel-V relationships for WT and mutant hERG channels that perturbed charge movement. B, bar graph plot of {Delta}{Delta}GQrel-V against primary amino acid sequence position. The box indicates the approximate limits of the S4 {alpha}-helix. The x-axis limits of the box indicate a {Delta}{Delta}GQrel-V of ±1 kcal/mol. A single value for |{Delta}{Delta}GQrel-V| could not be estimated for D540A and the more negative value is considered here (blue asterisk, Table I). C, frequency histogram plot of the number of substitutions that produced a given |{Delta}{Delta}GQrel-V| (D540A, blue asterisk). D, colorimetric representation of |{Delta}{Delta}GQrel-V| plotted onto a surface representation of the scanned region based on the KvAP isolated S1–S4 crystal structure (33). The residues are colored brighter blue to reflect greater perturbations in |{Delta}{Delta}GQrel-V|. The surface was made by rolling a 1.4 Å "ball" of water over the Van der Waals surface of the modeled segment. Asp540 is colored bright blue based on the |{Delta}{Delta}GQrel-V| calculated from the hyperpolarized component of a fit to a double Boltzmann function (Table I). Three orientations are shown, rotated clockwise by 0°, 120°, and 240° from left to right. A thread is formed by Arg531, Val535, Ala536, and Asp540 that turns around the S4 helix. Leu524 is located two helical turns above this ridge on the same side of the S4 {alpha}-helix. Residues in the linkers had little effect on charge movement. Significant residues are labeled to orient the reader.

 
The V0.5 and z values calculated from the Qrel-V relationships for WT and mutant channels (Table I) were used to estimate the {Delta}{Delta}GQrel-V. Fig. 3B plots the {Delta}{Delta}GQrel-V against the primary amino acid position for the entire Ala-scanned region. The box represents the approximate border of the S4 domain (y-axis limits) and {Delta}{Delta}GQrel-V values of ±1 kcal/mol (x-limits). A frequency histogram of perturbation magnitudes is plotted in Fig. 3C. Five mutations (L524A, R531A, V535A, A536V, and D540A) clearly perturbed the {Delta}{Delta}GQrel-V more than the other mutations, with values ≥ 1 kcal/mol. These mutations also segregated to a well defined subgroup when clustered using average linkage of the |{Delta}{Delta}GQrel-V| distances between groups (data not shown). Because the definition of a threshold to separate residues into two groups (non-perturbing and perturbing) creates an arbitrary bias, a linear color scale was constructed so that {Blue Brightness} {alpha} |{Delta}{Delta}GQrel-V|. On this scale, no perturbation is equated to the absence of blue (white), increasing degrees of perturbation (|{Delta}{Delta}GQrel-V|) are equated to an increase in the intensity of the color blue, and non-functional substitutions are colored gray. These color values were then mapped onto a helical surface representation of S4 (Fig. 3D) based on the isolated KvAP voltage sensor structure (33). As noted above, the Qrel-V relationship for D540A channels had two components. In the plot shown in Fig. 3D, the more negative values of z and V0.5 were used to estimate the {Delta}{Delta}GQrel-V value. Residues corresponding to the largest perturbations in |{Delta}{Delta}GQrel-V| (bright blue) mapped onto a spiral thread that turns around the length of S4, interrupted by the two non-expressing mutants K525A and L529A (Fig. 3D and Supplemental Movie 1).

Substitutions throughout the Scanned Region Perturbed Activation—Ionic currents were measured from each mutant channel in response to depolarizing steps, and grel-V relationships were constructed. Representative currents from WT and two mutant channels recorded at three test potentials are compared in Fig. 4. S517A channels activated at more positive potentials and deactivated more rapidly than WT (Fig. 4, A and B). K538A channel currents activated at more negative potentials and deactivated more slowly than WT currents (Fig. 4C). Although these and several other mutations altered the kinetics of current activation and deactivation, here we use alterations in the voltage dependence of activation to estimate perturbations induced by mutation. The V0.5, z, and {Delta}Ggrel-V values were determined from grel-V relationships and are summarized in Table II. The grel-V relationships for WT and ten mutant channels that perturbed the |{Delta}{Delta}Ggrel-V| ≥ 1 kcal/mol are plotted in Fig. 5A.



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  		FIG. 4.
Ionic currents elicited by depolarization of oocytes expressing WT and representative mutant channels that perturbed activation but not charge movement. A–C, currents elicited by steps to -20, 0, and +20 mV from a HP of -80 mV for WT (A), S517A (B), and K538A (C) hERG channels.

 



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  		FIG. 5.
Effects of Ala substitutions on the voltage dependence of activation. A, grel-V relationships for WT and mutant hERG channels that perturbed activation. B, bar graph plot of the {Delta}{Delta}Ggrel-V against primary amino acid sequence position. The box indicates the approximate limits of the S4 {alpha}-helix. The x-axis limits of the box indicate a {Delta}{Delta}Ggrel-V of ± 1 kcal/mol. C, frequency histogram plot of the number of positions that produced a given |{Delta}{Delta}Ggrel-V|. D, colorimetric representation of |{Delta}{Delta}Ggrel-V| plotted onto a surface representation of the scanned region as in Fig. 3D but with a brighter green color proportional to the magnitude of the effect on |{Delta}{Delta}Ggrel-V|. The exact same orientations used in Fig. 3D are replicated for comparison.

 
The estimated {Delta}{Delta}Ggrel-V was plotted against residue position in the primary amino acid sequence (Fig. 5B) and showed that substitutions that perturbed activation were located throughout the scanned region. Histogram analysis of the frequency of the |{Delta}{Delta}Ggrel-V| values caused by the mutations revealed a more continuous range of perturbation magnitudes than for the |{Delta}{Delta}GQrel-V| (Fig. 5C). However, a peak rising at ~1 kcal/mol separates a subset of ten mutations that perturbed the energetics of activation to a larger degree than most and segregated to a subgroup by average linkage cluster analysis on the distance between the |{Delta}{Delta}Ggrel-V| values. The |{Delta}{Delta}Ggrel-V| for four mutant channels (G516A, G522A, A527V, and R528A) that did not express well enough to permit gating current recordings was <1 kcal/mol. The five mutations that perturbed charge movement (L524A, R531A, V535A, A536V, and D540A) also significantly perturbed activation. R531A caused the largest perturbation in charge movement ({Delta}{Delta}GQrel-V = 1.9 kcal/mole) and activation ({Delta}{Delta}Ggrel-V = 4.3 kcal/mole). Five mutations perturbed |{Delta}{Delta}Ggrel-V| without altering the associated gating charge movement and were located in the S3–S4 linker (S517A and E518A), the lower-S4 (K538A), and the S4–S5 linker (Y542A and S543A). The dissociation between perturbation of charge movement and activation suggests that these residues might be involved in coupling voltage sensor movement to channel opening. Previous studies of Shaker and EAG channel activation concluded that specific basic residues in the S4 form electrostatic interactions with acidic residues in S2 and S3 (37). We have begun investigating these putative interactions in hERG and found that D456A causes a positive shift in the voltage dependence of activation ({Delta}{Delta}Ggrel-V = 2.67 ± 0.12 kcal/mol, n = 3) as does D460A ({Delta}{Delta}Ggrel-V = 2.76 ± 0.13 kcal/mol, n = 5). A green color scale corresponding to |{Delta}{Delta}Ggrel-V| was constructed, and the color values were mapped onto a helical surface representation of S4 (Fig. 5D) and displayed in the same three orientations as in Fig. 3D. Residues where Ala substitution caused large perturbations in the voltage dependence of activation (|{Delta}{Delta}Ggrel-V| ≥1 kcal/mol, bright green) were located throughout the scanned region. However, residues where substitution caused no discernable effect (white) localized to one face of the S4 helix (Fig. 5D, right panel, and Supplemental Movie 2).

Substitutions That Perturbed Inactivation Segregated to a Single Face of S4 —The voltage dependence of recovery from inactivation was assessed using a three-pulse protocol described under "Experimental Procedures." Representative currents recorded during the second pulse when channels recovered from inactivation and the third pulse when channels inactivated are shown in Fig. 6 for WT and two representative mutant channels that shifted the voltage dependence of recovery from inactivation in the depolarized (L523A, Fig. 6B) or hyperpolarized direction (A527V, Fig. 6C). The Iinact-V relationships were fitted by Boltzmann functions to estimate the V0.5, z, and {Delta}GIinact-V of inactivation (Table III). The Iinact-V relationships for WT and mutant channels that perturbed the {Delta}{Delta}GIinact-V are plotted in Fig. 7A. Mutation-induced perturbations in inactivation were less prominent than perturbations in charge movement or activation. Only one substitution, Y542A, perturbed the |{Delta}{Delta}GIinact-V| > 1 kcal/mol. However, nine of the substitutions had a |{Delta}{Delta}GIinact-V| of ≥0.5 kcal/mol (Fig. 7B) and were well separated from the other mutant channels by average linkage cluster analysis of the differences between |{Delta}{Delta}GIinact-V| values. Two of these nine inactivation-sensitive residues also perturbed activation (R531A and Y542A) and one (R531A) also perturbed charge movement associated with activation. A plot of the estimated {Delta}{Delta}GIinact-V versus residue position is plotted in Fig. 7C.



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  		FIG. 6.
Ionic currents recorded using the triple-pulse protocol to monitor recovery from inactivation. A–C, currents recorded during the interpulse and test pulse of the triple pulse protocol for WT (A), L523A (B), and A527V (C) mutant channels. The membrane potential was stepped to +40 mV for 300 ms to activate and inactivate channels, then repolarized through a range of potentials (in these examples, -120, -100, and -80 mV) for 10 ms to recover channels, and stepped back to +40 mV to assess the magnitude of recovered current. The currents recorded during the test (final) pulse are the largest following an interpulse potential of -120 mV. The voltage protocol is shown in the bottom panel where the timescale is expanded as shown by the dotted lines to better represent the recovery from and re-onset of inactivation.

 


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  		TABLE III
Inactivation parameters of hERG channels derived from Iinact-V relationships fitted with Boltzmann functions

 



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  		FIG. 7.
Effects of Ala substitutions on the voltage dependence of recovery from inactivation. A, Iinact-V relationships for WT and mutant hERG channels that perturbed inactivation. B, bar graph plot of the {Delta}{Delta}GIinact-V against primary amino acid sequence position. The box indicates the approximate limits of the S4 {alpha}-helix. The x-axis limits of the box indicate a {Delta}{Delta}GIinact-V of ± 0.5 kcal/mol. C, frequency histogram plot of the number of positions that produced a given |{Delta}{Delta}GIinact-V|. D, colorimetric representation of |{Delta}{Delta}GIinact-V| plotted onto a surface representation of the scanned region as in Fig. 3D but with a brighter red color proportional to the magnitude of the effect on |{Delta}{Delta}GIinact-V|. The exact same orientations used in Fig. 3D are replicated for comparison.

 
The range of |{Delta}{Delta}GIinact-V| was converted to a red scale and plotted onto a helical surface representation of S4 (Fig. 7D and Supplemental Movie 3). The residues that impacted the Iinact-V relationship are spaced ~3 residues apart and segregate to a stripe that spans the length of the S4 helix on one side. The inactivation-perturbing residues mapped to the same region that was nearly devoid of activation-perturbing residues (shown as white in Fig. 5D).

Helical Wheel Analysis—The results from each equilibrium distribution analysis are summarized as helical wheel plots in Fig. 8. Here, a coarser, albeit more arbitrary analysis, of the gradient of |{Delta}{Delta}G| was carried out by assigning residues to one of three groups (small effect, intermediate effect, and large effect). The 7 S3–S4 linker residues are represented in the inner ring, the 20 S4 residues as a middle ring of 18 and 2 in the inner ring, and the 5 S4–S5 linker residues as an outer ring. Residues where Ala substitution had the largest effects on activation or inactivation segregated to separate sides of the wheel. Residues where substitution perturbed charge movement are grouped within S4 (Fig. 8A). Conversely, the residues that perturbed activation, without altering charge movement, localized mainly to the S3–S4 and S4–S5 linkers (Fig. 8, A and B). The inactivation-sensitive residues clustered to one side of S4 (Fig. 8C) and were mainly exclusive of the activation-sensitive residues. A merged helical wheel plot (Fig. 8D) more clearly illustrates that the inactivation-sensitive residues define a face separate from the activation-sensitive residues.



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  		FIG. 8.
Helical wheel plots of Ala-scanned region highlighting perturbations in {Delta}{Delta}G caused by mutation of single residues. A, substitutions affecting the Qrel-V relationship. Residue positions where substitution perturbed the |{Delta}{Delta}GQrel-V| < 0.5 kcal/mol are colored white, 0.5 < |{Delta}{Delta}GQrel-V| < 1.0 kcal/mol are colored gray, and |{Delta}{Delta}GQrel-V| ≥ 1.0 kcal/mol are colored blue. B, substitutions affecting the grel-V relationship. Same coloring as in A except that positions where substitution caused a |{Delta}{Delta}Ggrel-V| ≥ 1.0 kcal/mol are colored green. C, substitutions affecting the Iinact-V relationship. Residue positions where substitutions perturbed the |{Delta}{Delta}GIinact-V| < 0.2 kcal/mol are colored white, 0.2 < |{Delta}{Delta}GIinact-V| < 0.5 kcal/mol are colored gray, and |{Delta}{Delta}GIinact-V| ≥ 0.5 kcal/mol are colored red. D, summary of perturbations plotted on a composite helical wheel. The colored residues from A–C were merged to create a composite plot. Residue positions where substitution perturbed the |{Delta}{Delta}GQrel-V| ≥ 1.0 kcal/mol also perturbed the |{Delta}{Delta}Ggrel-V| ≥ 1.0 kcal/mol and are colored cyan (Leu524, Val535, and Ala536). Y542A perturbed the |{Delta}{Delta}GIinact-V| ≥ 0.5 kcal/mol, and the |{Delta}{Delta}Ggrel-V| ≥ 1.0 kcal/mol and is colored orange. R531A affected all of the measured equilibrium distributions and is colored violet.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In a previous study we were somewhat surprised to have found no discernable difference between the gating current properties of WT and inactivation-deficient hERG channels (25). These findings provided the motivation to determine if more subtle measures of channel gating, ascertained by point mutation-induced perturbations in ionic and gating currents, might differentiate between activation and inactivation. This approach yielded some unexpected results. Ala substitutions that caused the greatest perturbations of charge movement followed a spiral groove along the S4 helix and, as expected, perturbed the voltage dependence of channel opening. Somewhat unexpectedly, Ala substitutions that perturbed activation were distributed throughout the scanned region, with the exception of one helical face of S4. Residues where substitution impacted inactivation formed a stripe along the helical face of S4 that corresponded to low impact activation positions. These findings suggest that the S4 domain acts as the voltage sensor for both activation and inactivation gating of hERG channels.

Interpretations and Limitations of the Ala Scan—The Ala scan analysis can only identify residues that experience different local energetics in one state compared with another state. If a residue moves or is repacked in the open versus the closed state but the local energy remains similar, no perturbation will be observed. This analysis cannot differentiate between residues that have not moved and residues that have moved, but into a similar energetic environment (e.g. from one hydrophobic pocket to another, or from one aqueous environment to another). Smaller state-dependent differences might be amplified by substituting a different amino acid that would cause larger perturbations by introducing volume, hydrophobicity, charge, polarity, or non-polarity. However, because Ala is expected to have minimal physical interactions with nearby residues and occupies a relatively flexible Phi-Psi space, Ala substitution should alter the WT residue functionality with the least amount of distortion compared with substitution with another amino acid.

Inherent in the equilibrium analysis is the assumption that gating transitions can be simplified by considering only two states (e.g. closed versus open). Obviously, these assumptions oversimplify a complicated sequence of events that are likely to involve many channel states. Nonetheless, because the equilibrium distributions measured (Qrel-V, grel-V, Iinact-V) can be relatively well fit by single Boltzmann functions, simplified two-state models have been commonly used to analyze the impact of substitutions on steady-state gating properties for other K+ channels (16, 38–42). Although shifts in the V0.5 can be used to assess perturbation, it does not address the valence or steepness of the distribution. By estimating {Delta}G, both the steepness (z) and midpoint of the distribution (V0.5) are taken into account.

Cluster analysis was used to define threshold values for |{Delta}{Delta}G| considered to represent significant mutation-induced perturbation in channel gating. Using this method, the threshold value of |{Delta}{Delta}G| for recovery from inactivation was lower than for the Qrel-V or grel-V relationships, suggesting that single S4 residue interactions do not contribute as much energetically to channel inactivation as they do to activation. Recovery from inactivation may be stabilized by multiple, lower energy residue interactions that involve the one surface identified by this scan. The valence associated with inactivation (z = 1) was lower than the valence associated with activation gating (z = 3). Because |{Delta}{Delta}G| = zFV, this difference also contributed to the lower values of |{Delta}{Delta}G| calculated for inactivation compared with activation. Finally, it should be noted that we have made no attempt to differentiate between the different mechanisms that result in a negative versus a positive value in {Delta}{Delta}G. For example, a negative value of {Delta}{Delta}G for activation could result from a mutation-induced stabilization of the open state or a destabilization of the closed state.

Comparisons with Other Large Scale Scanning Mutagenesis Studies—Scanning mutagenesis studies of the S4 domain in other Kv channels have identified high impact residues that participate in, or sense rearrangements associated with channel activation or inactivation. Li-Smerin and colleagues (16) performed a systematic Ala scan of the entire S1–S4 domain in Kv2.1. In their study, 13 residues within S4 and the flanking linkers perturbed |{Delta}{Delta}Ggrel-V| > 1 kcal/mol. Nearly all (9/10) of the activation-sensitive hERG residues identified in our study overlapped with the activation-sensitive Kv2.1 residues, in good agreement with a general role for these residues in charge movement or coupling charge movement to channel opening. A more limited scan of the hERG S4 was recently reported by Subbiah et al. (43) who substituted Gln for the basic residues in S4 (Lys525, Arg528, Arg531, Arg534, and Arg537). They found that each substitution, with the exception of R534Q, perturbed activation; however, effects on inactivation were not evaluated. In contrast, we found that R528A, R534A, and R537A did not appreciably perturb activation (but did perturb inactivation). These differences are likely due to the fact that Ala substitution of a basic residue completely removes charge and polarity at that position, whereas Gln substitution retains polarity and may introduce non-native interactions compared with Ala. A scan of Shaker S4 using Cys-labeled fluorophores identified residues that experienced fluorescence changes during activation, slow inactivation, or both processes (44). This study suggested that a large group of fluorophore-labeled S4 residues pointing in all directions sense changes in a local environment that accompany S4 entry into the slow inactivation conformation. However, the residues in Shaker that reported on inactivation alone were found to reside in the mid-portion of S4 and correspond to Kv2.1 and hERG residues that dramatically altered activation gating. The marked differences between these studies are difficult to reconcile but may be due to the underlying differences in methodology ({Delta}{Delta}G versus kinetics of change in fluorescence).

Putative Interactions and Coupling in the Activation and Inactivation Pathways—We mapped the gating-sensitive residues onto a Kv channel structural model (34) to examine potential inter-helical contacts. This Shaker channel model was constructed using the open MthK channel (45, 46) with the S1–S4 domains constrained by a series of experimental observations, including interactions between S4 and specific regions in the S2, S3, S5, and S6 domains (34). Although based mainly on functional data, this homology model provides a reasonable framework to predict potential interactions between the S4 and neighboring domains that mediate hERG channel activation and inactivation. We also mapped the charge- and inactivation-perturbing residues onto a homology model constructed from the isolated (S1–S4) KvAP voltage sensor (33) (data not shown). However, because the precise orientation of S4 with respect to S1–S3 and S5–S6 remains unclear, the KvAP model was not a useful tool to predict how the gating-sensitive residues might interact with neighboring helices.

Residues causing the greatest perturbations in charge movement formed a spiral around S4, roughly defining a thread along a helical screw. The continuity of the thread was interrupted by our inability to record currents from two residues (Lys525 and Leu529) that were intolerant to Ala substitution. The observation that charge-perturbing residues follow a helical groove is consistent with previous hypotheses that membrane depolarization induces a helical twist or rotation of the S4 domain that translocates charge across the electrical field (34, 47–49). Our findings also suggest a critical role for Arg531 in hERG voltage sensor movement, because Ala substitution caused the largest perturbation among all the scanned residues. Our study was not designed to identify specific residues in neighboring domains that might influence depolarization-induce S4 movement. However, based on the proposed interactions between acidic S2 residues and basic S4 residues in Shaker and EAG channels (34, 37, 50–54), the shifts in activation produced by Ala substitution of Asp residues in hERG S2 in this study and Cys substitution by Liu et al. (55), and the proximity of Asp456 and Asp460 to Arg531 in the hERG homology model (Fig. 9, <3 Å), we speculate that electrostatic interactions between these Asp residues and Arg531 may stabilize the activated state of the voltage sensor.



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  		FIG. 9.
Structural model for hERG helical packing suggests specific interactions of S4 with surrounding domains. Homology model of hERG S1–S6 based on an open Shaker channel model (34). S4 is represented as a surface (residues colored as in Fig. 8D), and neighboring helices are represented as ribbons: S1 (pink), S2 (orange), S3 (yellow), S5 (blue), and S6 (violet). A, S2 forms a putative interaction surface with the charge-sensitive residues in S4. B, N-terminal S5 residues may interact with inactivation-sensitive residues in S4.

 
Residues in the S3–S4 linker, S4–S5 linker, and C-terminal end of S4 perturbed activation without markedly altering charge movement. Thus, these residues likely couple movement of S4 to the activation gate. A role for the S3–S4 linker in stabilizing the open channel state is supported by disulfide cross-linking and electrostatic studies in Shaker indicating that the linker approaches the extracellular end of S5 in the open channel state (34, 47, 56–59). An important role for the S4–S5 linker in coupling voltage sensor movement to the activation gate has been proposed for Shaker (60), a K+ channel from Arabidopsis thaliana, KAT1 (61), hERG (62, 63), and hyperpolarization activated cyclic nucleotide-gated cation channels (64, 65). Because the S4–S5 linker is tethered to the S4 domain and connected to the pore via S5, depolarization-induced S4 movement may be transduced via the linker to opening of the activation gate.

How is movement of the voltage sensor coupled to inactivation in hERG channels? The channel configuration and S4 movement associated with recovery from inactivation is markedly different than that associated with activation. Consistent with this idea, mutation-induced perturbations in hERG inactivation mapped to a face of S4 distinct from the activation-sensitive residues. The inactivation-sensitive subset of residues was distributed across the entire length of the S4 helix, indicating that the whole surface experiences a rearrangement associated with inactivation. A small rigid body tilt of S4 would be consistent with the longitudinal distribution of mutation-induced perturbations in inactivation. The Laine et al. model suggests the inactivation-sensitive residues at the N-terminal end of the stripe (i.e. L523A, A527V, and R531A) are within 3 Å of S2, whereas residues at the C-terminal end (i.e. R537A, R541A, and E544A) are within 3 Å of S5 (Fig. 9 and Supplemental Movie 4). Although speculative, a small tilt of S4 could be stabilized by interactions with S2 and cause a rearrangement in the S5 domain that is transduced to the pore to close the inactivation gate. In this respect hERG inactivation may be different from Shaker, where coupling of the voltage sensor to C-type inactivation is believed to occur through discreet interactions between the N-terminal portion of S4 and the turret linking S5 to the pore helix (57, 58, 66, 67). Of note, the conserved Glu residue (Glu418) at the extracellular end of the Shaker S5 has been proposed to couple S4 to C-type inactivation is a Gly in hERG and EAG channels. The relatively small changes in {Delta}{Delta}GIinact-V values imply that the contribution of any single residue in S4 to the free energy of rearrangements associated with closure of the inactivation gate is relatively small compared with activation, and is consistent with a distributed patch of inactivation-sensitive S4 residues making multiple contacts with surrounding domains.

Summary—Our study identified residues in the S4 helix and flanking linkers of hERG that experienced repacking or strain during rearrangements associated with movement of the voltage sensor and opening of the activation or inactivation gates. Ala substitutions that caused the greatest perturbations of charge movement localized to a spiral thread along the S4 helix, consistent with a depolarization-induced helical twist or rotation of S4. Mutations that perturbed activation were distributed throughout the scanned region, with the exception of one helical face of S4. Residues that impacted inactivation formed a stripe along the helical face of S4 that corresponded to low impact activation positions. Taken together, these results suggest that the S4 domain acts as a voltage sensor for both activation and inactivation gating of hERG channels. Unlike Shaker channels, where discreet interactions between S4 and the pore helix couple voltage sensor movement to C-type inactivation, an entire helical face of S4 may be important in the voltage-sensing mechanism that drives hERG inactivation.


   FOOTNOTES
 
* This work was supported by NHLBI, National Institutes of Health Grants HL65299 (to M. C. S.) and HL03816 and HL75536 (to M. T.-F.) and by American Heart Association fellowships (to D. R. P. and W. A. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Supplemental Movies 1-4. Back

To whom correspondence should be addressed: Dept. of Physiology, Nora Eccles Harrison CVRTI, University of Utah, 95 South 2000 East, Salt Lake City, UT 84112. Tel.: 801-585-3682; Fax: 801-581-3128; E-mail: piper{at}cvrti.utah.edu.

1 The abbreviations used are: hERG, human ether-a'-go-go-related gene; Kv, voltage-gated K+; WT, wild-type; EAG, ether-a'-go-go; COVG, cut-open Vaseline gap; HP, holding potential; IgOFF, off-gating current; IgON, on-gating current; Qrel-V, relative charge-voltage; grel-V, relative conductance-voltage; Iinact-V, relative current recovered from inactivation-voltage; MES, 2-(N-morpholino)ethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank Krista Kinard, Meng San Pun, and Kam Hoe for technical assistance.



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 DISCUSSION
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P. Ju, G. Pages, R. P. Riek, P.-c. Chen, A. M. Torres, P. S. Bansal, S. Kuyucak, P. W. Kuchel, and J. I. Vandenberg
The Pore Domain Outer Helix Contributes to Both Activation and Inactivation of the hERG K+ Channel
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G. Panaghie and G. W. Abbott
The Role of S4 Charges in Voltage-dependent and Voltage-independent KCNQ1 Potassium Channel Complexes
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J. I. Vandenberg, A. Varghese, Y. Lu, J. A. Bursill, M. P. Mahaut-Smith, and C. L.-H. Huang
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T. Ferrer, J. Rupp, D. R. Piper, and M. Tristani-Firouzi
The S4-S5 Linker Directly Couples Voltage Sensor Movement to the Activation Gate in the Human Ether-a-go-go-related Gene (hERG) K+ Channel
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T. S. Surti, L. Huang, Y. N. Jan, L. Y. Jan, and E. C. Cooper
Identification by mass spectrometry and functional characterization of two phosphorylation sites of KCNQ2/KCNQ3 channels
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R. N. Subbiah, M. Kondo, T. J. Campbell, and J. I. Vandenberg
Tryptophan scanning mutagenesis of the HERG K+ channel: the S4 domain is loosely packed and likely to be lipid exposed
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D. Fernandez, A. Ghanta, K. I Kinard, and M. C Sanguinetti
Molecular mapping of a site for Cd2+-induced modification of human ether-a-go-go-related gene (hERG) channel activation
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